U.S. patent number 7,087,395 [Application Number 09/761,969] was granted by the patent office on 2006-08-08 for vitamin d assay.
This patent grant is currently assigned to Quest Diagnostics Investments Incorporated. Invention is credited to Martha Garrity, Jacqueline Tran.
United States Patent |
7,087,395 |
Garrity , et al. |
August 8, 2006 |
Vitamin D assay
Abstract
The present invention features a kit and a method of using the
kit for determining a concentration of a vitamin D component. The
kit comprises a releasing composition and a detecting composition.
The releasing composition comprises an aqueous base component. In
one embodiment, the releasing composition is substantially free
from an organic solvent.
Inventors: |
Garrity; Martha (San Clemente,
CA), Tran; Jacqueline (Westminster, CA) |
Assignee: |
Quest Diagnostics Investments
Incorporated (Wilmington, DE)
|
Family
ID: |
25063750 |
Appl.
No.: |
09/761,969 |
Filed: |
January 16, 2001 |
Current U.S.
Class: |
435/7.93;
422/417; 424/1.49; 435/7.1; 435/7.9; 435/7.92; 436/501; 436/518;
436/536; 436/542; 536/23.1; 536/24.3; 536/24.33 |
Current CPC
Class: |
G01N
33/82 (20130101) |
Current International
Class: |
G01N
33/53 (20060101); G01N 21/00 (20060101); G01N
33/543 (20060101) |
Field of
Search: |
;436/501,536,542,518
;424/1.49 ;435/6,7.1-7.9,91.2,7.92,7.93 ;536/23.1,24.3,24.33
;422/6,1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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198 40 435 |
|
Dec 1999 |
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DE |
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0 750 743 |
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Jan 1997 |
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EP |
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1392403 |
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Apr 1975 |
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GB |
|
WO89/05356 |
|
Jun 1989 |
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WO |
|
Other References
Kobayashi et al., "Evaluation of solubilizing agents for
25-hydroxy-vitamin D3 immunoassay." Clinica Chimica Acta, 1992, pp.
83-88. cited by examiner .
Aldrich Catalogue, 1992, pp. 1949-1950. cited by examiner .
"Measurement of Calcium, Phosphate, Parathyroid Hormone, and
Vitamin D" by Heinrich Schmidt-Gayk; Dynamics of Bone and Cartilage
Metabolism pp. 375-399; (1999). cited by other .
"Determination of vitamin D3 metabolites: state-of-the-art and
trends" by Luque de Castro et al.; Journal of Pharmaceutical and
Biomedical Analysis 20 (1999) pp. 1-17. cited by other .
"Two Direct (Nonchromatographic) Assays for 25-Hydroxyvitamin D" by
Bouillon et al.; Clinical Chemistry, vol. 30 No. 11 pp 1731-1736;
(1984). cited by other .
Directional insert of Biosource International Europe S.A. entitled:
"OH-VIT.D3-RIA-CT". cited by other .
Directional insert of IDS, Inc. entitled: "Gamma-B 25-Hydroxy
Vitamin D RIA" AA-35PS, Jan. 6, 1999, issue 3. cited by other .
Directional insert of Nichols Institute Diagnostics entitled:
"25-OH-D 60T kit". cited by other .
Instruction Manual of Incstar Corporation (Jul. 1992). cited by
other .
Instruction manual for Jummn Diagnostik entitled: "25OHVitamin D
Enzume-based-Protein-Binding-Assay". cited by other .
Competitive protien-Binding Radioassays for 25-OH-D; Clinical
Applications; by Hadda, Jr. cited by other .
"The Simultaneous Measurement of Vitamin D Metabolites in Plasma:
Studies in Healthy Adults and in Patients with Calcium
Nephrolithiasis" by Caldas, A.E., et al.; Journal of Laboratory and
Clinical Medicine, St. Louis, MO, vol. 91, No. 5, (1978) pp.
840-849. cited by other .
"A Direct Non-Extraction Enzyme Immunoassay for Measurement of 25
Hydroxyvitamin D" by Gardner, M.J., et al.; Journal of Bone and
Mineral Research, New York, NY., vol. 16, No. Suppl 1, Sep. 2001 p.
S434, ANSU528. cited by other .
"Comparison of the Nichols Advantage and the Conventional
Competitive Protein Binding Assay for the Measurement of
Circulating Concentration of 25-Hydroxyvitamin D in Human and
Non-Human Primates." by Chen, T.C., et al.; Journal of Bone and
Mineral Research, vol. 16, No. Suppl 1; Sep. 2001, p. S434. cited
by other .
PCT International Search Report; Jun. 6, 2003. cited by
other.
|
Primary Examiner: Le; Long V.
Assistant Examiner: Cook; Lisa V.
Attorney, Agent or Firm: Stout, Uxa, Buyan & Mullins,
LLP Stout; Donald E. Hollrigel; Greg S.
Claims
What is claimed is:
1. A kit for determining a concentration of 25-hydroxy vitamin D
(25-OH-D) in a sample, comprising: a releasing composition
including from about 0.01 to about 5% of a cyclodextrin, from about
0.01 to about 5% of a sodium salicylate, and from about 0.1 to
about 1.0 M NaOH, the cyclodextrin, the salicylate, and the NaOH
being provided in an amount effective to reduce interference from a
protein or a lipid with 25-hydroxy vitamin D present in a sample;
25-hydroxy vitamin D coupled to a solid phase; vitamin D binding
protein; and a vitamin D binding protein antibody coupled to a
label present in an amount that produces a detectable signal when
25-hydroxy vitamin D is present in the sample.
2. A kit of claim 1, wherein the cyclodextrin, the salicylate, and
the NaOH are provided in an amount effective to reduce interference
from proteins or lipids with 25-hydroxy vitamin D present in a
sample of a mammal fluid.
3. A kit of claim 2 wherein the mammal fluid is selected from the
group consisting of milk whole blood, serum, and plasma.
4. A kit of claim 2 wherein the mammal fluid comprises a human
serum.
5. A kit of claim 1 wherein the releasing composition comprises
about 0.35 to about 0.5 M of NaOH.
6. A kit of claim 1 wherein the releasing composition is free of an
organic solvent.
7. A kit of claim 1 wherein the cyclodextrin is selected from the
group consisting of alpha-cyclodextrin and a beta-methylated
cyclodextrin.
8. A kit of claim 1 wherein the releasing composition comprises
about 2% of an alpha-cyclodextrin.
9. A kit of claim 1 wherein the releasing composition comprises
about 0.05% of a beta-methylated cyclodextrin.
10. A kit of claim 1 wherein the releasing composition comprises
about 0.5 to about 5% of the sodium salicylate.
11. A kit of claim 1 wherein the releasing composition further
comprises about 0.01 to about 0.1% of a surfactant.
12. A kit of claim 1 wherein the releasing composition forms a
homogeneous mixture with a mammal fluid.
13. A kit of claim 1 wherein the label is selected from the group
consisting of a chemiluminescent label, a fluorescent label and a
radio-active label.
14. A kit of claim 1 wherein the vitamin D binding protein antibody
is an acridinium-labeled antibody.
15. A kit of claim 1 wherein the solid phase is a magnetic
particle.
16. A kit of claim 1 wherein the 25-hydroxy vitamin D coupled to a
solid phase is in the form of 25-hydroxy vitamin D coated magnetic
particles, the vitamin D binding protein antibody is coupled to an
acridinium label, and the 25-hydroxy vitamin D coated magnetic
particles, the acridinium labeled vitamin D binding protein
antibody, and the vitamin D binding protein are present in a single
composition.
17. A kit of claim 1, wherein the releasing composition further
includes from about 0.01% to about 0.1% of a surfactant, and the
antibody is labeled with acridinium.
Description
BACKGROUND OF THE INVENTION
In 1928 Adolf Windaus was awarded the Nobel Prize in chemistry for
his research on substances of significant biological importance.
One of these substances was Vitamin D. Since then, much more is
known about vitamin D, its chemistry and its pharmacological
effects and dynamics.
Vitamin D is not widely found in food, but rather, it is produced
by the skin. Once produced, it undergoes 25-hydroxylation to form a
25-hydroxy-D (25-OH-D) in the liver. The circulating concentration
of 25-OH-D is considered to be an important indicator of vitamin D
status in man. For example, hypovitaminosis, which results from the
insufficient endogenous production of vitamin D in the skin, and
insufficient dietary supplementation, and/or inability of the small
intestine to absorb adequate amounts of vitamin D from dietary
sources, results in hypocalcemia and hypophosphatemia and
corresponding secondary hyperparathyroidism. Hypovitaminosis D also
results in disturbances in mineral metabolism (i.e., rickets and
osteomalacia in children and adults, respectively).
Serum 25-OH-D-levels are also found to be lower than normal in
intestinal malabsorption syndromes, liver disorders (chronic and
acute), and nephrotic syndromes. In osteopenia, especially in the
aged, serum 25-OH-D levels are often found to be lower than normal.
In cases of vitamin D intoxication, serum 25-OH-D level is found,
as expected, to be higher than normal.
Once hydroxylated, 25-OH-D is again hydroxylated in the kidney to
give the hormonal form 1,25-dihydroxy-vitamin D
(1,25-(OH).sub.2-D). The 1,25-(OH).sub.2-D level in blood is also
an important indicator of certain diseases. For example, a low
level of 1,25-(OH).sub.2-D is indicative of kidney failure and/or
osteoporosis.
Considering their pathological importance, tremendous efforts have
been directed towards developing assays for accurately measuring
concentrations of 25-OH-D and/or 1,25-(OH).sub.2-D in
circulation.
For example, 25-OH-D concentrations in blood have been measured by
high performance liquid chromatography (HPLC) and by competitive
protein binding assays (Eisman et al., Anal. Biochem. 80: 298 305
(1977); and Haddad et al., J. Clin. Endocr. 33: 992 995 (1971)).
For example, the vitamin D transport protein known as DBP, which
has a strong preference for binding 25-OH-D was used in the
competitive binding assay (Bouillion et al., J. Steroid Biochem.
13: 1029 1034 (1980)).
Also, various competitive binding assays have been employed to
assay for the presence of 1,25-(OH).sub.2-D. (Shigeharu et al.,
Anal. Biochem. 116: 211 222 (1981); Eisman et al., Arch. Biochem.
Biophys. 176: 235 243 (1976); Perry et al., Biochem. Biophys. Res.
Comm. 112: 431 436 (1983); Bouillion et al., Ann. Endocrin. 41: 435
436 (1980); Bouillion, Clin. Chem. 26: 562 567 (1980); Bouillion,
Eur. J. Biochem. 66: 285 291 (1976)). In such assays, vitamin D and
its metabolites were extracted from blood serum or plasma with an
organic solvent. The extract was then purified by column
chromatography and HPLC to yield 1,25-(OH).sub.2-D. The purified
1,25-(OH).sub.2-D was then measured.
In general, most assays presently being employed in the art to
determine the concentration of vitamin D are heterogeneous assays.
Furthermore, most of these assays rely on the addition of an
organic solvent to release the vitamin D and/or vitamin D
metabolites from the binding proteins, for example DBP.
(Schmidt-Gayk, Dynamics of Bone and Cartilage Metabolism, Chapter
26, Table V). That is, the addition of organic solvent to the
samples causes denaturation and precipitation of the serum proteins
including DBP, and subsequently, the precipitated protein can be
spun out of solution and the released metabolites remain in
solution in the organic layer. This organic layer containing the
released metabolite is then extracted and transferred into another
system for analysis.
Although the present existing assay systems are useful, their
reliance on an organic solvent to release the vitamin D and/or
vitamin D metabolite is problematic. The problems with using an
organic solvent include: (A) difficulties in the handling of
volatile organic solvents, (B) laborious manual extractions, (C)
loss of patient, identification since two transfer steps are
required, and (D) loss of precision in measurement.
The loss in precision may be caused by the heterogeneous extraction
step, since heterogeneous extraction can be very technique
dependent. For example, in a heterogeneous extraction step, some
matrix components may be extracted along with the organic layer
containing the metabolite. One such matrix component is lipid,
which has been shown to cause an elevated measurement of the
metabolite values.
There is a need to have improved kits and methods for determining
the concentration of vitamin D and its metabolites in a body fluid
of a mammal.
SUMMARY OF THE INVENTION
In accordance with the present invention, a kit is featured for
determining the concentration of a vitamin D component. In a broad
embodiment, the kit comprises a releasing composition. The
releasing composition facilitates in releasing the vitamin D
component from a vitamin D component binding-protein. In one
embodiment, the releasing composition is substantially free of an
organic solvent. In one embodiment, the kit further comprises a
detecting composition. The detecting composition facilitates in
determining the concentration of the vitamin D component.
Further in accordance with the present invention, a kit according
to this invention may be useful for determining the concentration
of the vitamin D component present in a mammal fluid. The mammal
fluid may be milk, whole blood, serum, plasma and mixtures thereof.
For example, a mammal fluid may comprise a human serum.
Still further in accordance with the present invention, the vitamin
D component may be a vitamin D, a precursor of a vitamin D, a
metabolite of a vitamin D and mixtures thereof. For example, a
vitamin D component comprises a 25-OH-D.
Still further in accordance with the present invention, the
releasing composition comprises an aqueous base component, for
example, NaOH or KOH. In one embodiment, the releasing composition
comprises about 0.1 to about 1.0 M of the aqueous base component.
In one embodiment, the releasing composition is substantially free
of an organic solvent. In one embodiment, the releasing composition
may be adapted to various bio-assay systems to assay for a vitamin
D component.
Still further in accordance with the present invention, the
releasing composition further comprises a cyclo-oligomer component,
for example a cyclodextrin, derivatives thereof and mixtures
thereof. In one embodiment, the releasing composition comprises
about 0.01 to about 5% of the cyclo-oligomer component.
Still further in accordance with the present invention, the
releasing component further comprises about 0.5 to about 5% of a
metal salicylate, including sodium salicylate.
Still further in accordance with the present invention, the
releasing component further comprises about 0.01 to about 0.1% of a
surfactant.
Still further in accordance with the present invention, the
releasing composition forms a homogeneous mixture with a mammal
fluid.
Still further in accordance with the present invention, the
detecting composition comprises a host component and a partner
component, wherein the host component binds to the partner
component to form a partner/host complex. The concentration of the
complex should be proportional to the concentration of the vitamin
D component. In one embodiment, the concentration of the complex is
inversely proportional to is the concentration of the vitamin D
component. In one embodiment, the host component comprises an
antibody and/or portions thereof.
Still further in accordance with the present invention, the host
component is labeled. In one embodiment, the host component is
labeled with a chemiluminescent label, for example acridinium, a
fluorescent label and/or a radio-active label.
Still further in accordance with the present invention, the partner
component comprises a vitamin D component linked to a separator
component, wherein the separator component is a solid phase, such
as antibody or a magnetic particle.
Still further in accordance with the present invention, the partner
component binds to the host component through at least one
intermediate binding component, for example, a vitamin D
binding-protein.
Still further in accordance with the present invention, a method is
featured for assaying a body fluid sample for the concentration of
a vitamin D component. The method comprises the steps of releasing
the vitamin D component from the vitamin D component
binding-protein by contacting the sample with a releasing
composition in a holder, and determining the concentration of the
vitamin D component.
Still further in accordance with the present invention, the
determining step includes the steps of (a) adding a detecting
composition to the holder, the detecting composition comprises a
host component and a partner component, the host component binds to
the partner component to form a partner/host complex, (b) isolate
the complex in the tube, (c) measuring the concentration of the
complex by measuring the concentration of the host component in the
complex, the concentration of the complex is proportional to the
concentration of the vitamin D component.
Any feature or combination of features described herein are
included within the scope of the present invention provided that
the features included in any such combination are not mutually
inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is an illustration of general assays (A through H) in which
a releasing composition may be employed.
FIG. 1B is a legend of the elements of FIG. 1A.
Additional advantages and aspects of the present invention are
apparent in the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
In a broad embodiment, the present invention provides a kit for
determining the concentration of a vitamin D component. In one
embodiment, the kit is useful for determining the concentration of
a vitamin D component found in a mammal, for example a human. In a
preferred embodiment, the kit is useful for determining the
concentration of a vitamin D component in a body fluid of a mammal.
The body fluid may include, without limitation, milk, whole blood,
serum, plasma or mixtures thereof. Preferably, the body fluid is
serum of a human.
A vitamin D component includes a vitamin D, vitamin D precursors
and vitamin metabolites. Without intending to be limited by the
examples, a vitamin D component may include, for example, vitamin
D.sub.2, D.sub.3, D.sub.4, D.sub.5, and D.sub.6; precursors of a
vitamin D such as 7-dehydrocholesterol; and metabolites of vitamin
D such as metabolites of vitamin D.sub.2, D.sub.3, D.sub.4,
D.sub.5, and D.sub.6, preferably 1,25-OH-D.sub.2, and more
preferably 25-OH-D. Often, a vitamin D component is bound to a
vitamin D component binding protein. For example, in human serum,
25-OH-D is bound to a protein; this protein is commonly referred to
in the art as a DBP.
A kit of the present invention comprises a releasing composition.
The releasing composition of this invention is adaptable for use in
or with various bio-assay techniques known in the art to determine
the concentration of a vitamin D component. Preferably, the
releasing composition of the present invention substantially
eliminates the need to purify the vitamin D component prior to
determining its concentration. Also preferably, a bio-assay kit
comprising a releasing composition of this invention has improved
precision in the measurement of a vitamin D component.
In one embodiment, the releasing composition comprises an aqueous
base component and facilitates in releasing the vitamin D component
from a binding entity. The binding entity includes, without
limitation, a binding protein or a binding lipid. The aqueous base
component comprises any molecule which is effective in raising and
maintaining the pH of the releasing composition to a pH of greater
than 7, preferably greater than 8, more preferably about 12 to
about 14, even more preferably about 13. In one embodiment, the
base component comprises a metal hydroxide, for example a NaOH
and/or a KOH. In one embodiment, the releasing composition is
substantially free of an organic solvent, for example
acetonitrile.
In a preferred embodiment, the releasing composition comprises
about 0.1 to about 1 M of the aqueous base component. More
preferably, the releasing composition comprises about 0.35 to about
0.5 M of the aqueous base component. For example, a releasing
composition in accordance with the present invention may include
about 0.35 to about 0.5 M of NaOH.
Without wishing to limit the invention to any particular theory or
mechanism of operation, it is believed that the aqueous base
component denatures, preferably irreversibly denatures, the vitamin
D binding protein, i.e. DBP, and causes the bound vitamin D
component to be released. Additionally, the aqueous base component
may also irreversibly denature other plasma or serum proteins, such
as albumin. It is further believed that these proteins may
interfere in the detection of the vitamin D component and their
denaturation reduces this effect.
In one embodiment, the releasing composition further comprises a
cyclo-oligomer component. In a preferred embodiment, the
cyclo-oligomer component is at least effective in sequestering
interfering components. Interfering components, without limitation,
include hydrophobic molecules, such as lipids. Interfering
components interfere with the measurement of the vitamin D
component, and therefore, its sequestration substantially reduces
interference of the measurement. For example, lipids have been
shown to erroneously increase the measurement values of a vitamin D
component.
In a preferred embodiment, the cyclo-oligomer component comprises
an .alpha.-cyclodextrin. In a more preferred embodiment, the
cyclo-oligomer component comprises a .beta.-randomly methylated
cyclodextrin. The cyclo-oligomer component may also include,
without limitation, derivatives of .alpha.-cyclodextrin,
.beta.-cyclodextrin, derivatives of .beta.-cyclodextrin,
.gamma.-cyclodextrin, derivatives of .gamma.-cyclodextrin,
carboxymethyl-.beta.-cyclodextrin,
carboxymethyl-ethyl-.beta.-cyclodextrin,
diethyl-.beta.-cyclodextrin, dimethyl-.beta.-cyclodextrin,
methyl-.beta.-cyclodextrin, random methyl-.beta.-cyclodextrin,
glucosyl-.beta.-cyclodextrin, maltosyl-.beta.-cyclodextrin,
hydroxyethyl-.beta.-cyclodextrin,
hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin, and the like and mixtures
thereof. As used herein, the term "derivatives" as it relates to a
cyclodextrin means any substituted or otherwise modified compound,
which has the characteristic chemical structure of the parent
cyclodextrin.
In one embodiment, the releasing composition comprises about 0.01
to about 5% of the cyclo-oligomer component. For example, in a
preferred embodiment, the releasing composition comprises about 2%
of an .alpha.-cyclodextrin. In another preferred embodiment, the
releasing composition comprises about 0.05% of a .beta.-randomly
methylated cyclodextrin. In an alternative embodiment, the
releasing composition is substantially free of a cyclo-oligomer
component.
In one embodiment, the releasing component further comprises a
salicylate and the like, such as 8-anilino-1-naphthalenesulfonic
acid (ANS). Without wishing to be limited by any theory or
mechanism of operation, it is believed that the salicylate or the
like of the present invention is at least effective to release the
vitamin D component from binding to proteins and/or solubilize the
vitamin D component in the releasing composition. In a preferred
embodiment, the salicylate is a metal salicylate, for example
sodium salicylate. The release composition preferably comprises
about 0.5 to about 5% of a metal salicylate, for example a sodium
salicylate. In an alternative embodiment, the releasing composition
is substantially free of a salicylate and the like.
In one embodiment, the releasing component further comprises a
surfactant and the like. Without wishing to be limited by any
theory or mechanism of operation, it is believed that the
surfactant and the like of the present invention is at least
effective to block the vitamin D component from being attached to
lipids, proteins and the like. In a preferred embodiment, the
surfactant is TWEEN-20 or TRITON X-100. The releasing composition
of the present invention may include about 0.01 to about 0.1% of a
surfactant. For example, 0.05% of TWEEN-20 may preferably be
employed in the present invention. In another preferred embodiment,
the releasing component is substantially free of a surfactant.
The releasing composition of the present invention may comprise an
aqueous base component and may be substantially free of the
cyclo-oligomer component, the salicylate and the surfactant. In a
preferred embodiment, the release component comprises an aqueous
base component and at least a cyclo-oligomer component, a
salicylate and/or a surfactant. In a more preferred embodiment, the
release component comprises an aqueous base component and a
cyclo-oligomer component. In an even more preferred embodiment, the
release component comprises an aqueous base component, a
cyclo-oligomer component and a salicylate. For example, a preferred
releasing composition comprises about 0.1 to about 1 M of an
aqueous base component, about 0.01 to about 5% of a cyclo-oligomer
component and about 0.01 to about 5% of a salicylate.
As discussed above, the releasing composition of this invention is
adaptable for use in or with various bio-assay techniques known in
the art to determine the concentration of a vitamin D component.
For example, in one embodiment, a kit of the present invention
comprises a releasing composition and a detecting composition.
In a broad embodiment, the detecting composition of the present
invention comprises a host component. Preferably, the detecting
composition further comprises a partner component. The host
component is any molecule or set of molecules capable of
selectively binding to a partner component or a vitamin D
component. For example, a host component may be an aptamer or a
molecular imprint polymer. In one embodiment, a host component is
an antibody, or a portion of an antibody, for example a Fab
portion, capable of binding to a partner component. The antibody of
the present invention may be monoclonal or polyclonal.
Various methods are known in the art to produce an antibody
specific toward a certain antigen, for example a partner component.
For example, an antibody may be raised from a rabbit injected with
an antigen, the antigen being the partner component or a part
thereof. Additionally, synthetic antibodies may also be made. (See
U.S. Pat. No. 5,110,833, the disclosure of which is incorporated in
its entirety by reference herein.)
The specific composition of the partner component depends on the
nature of the assay. For example, if a competitive assay technique
is employed, the partner component may comprise a competing
molecule which is capable of competing with the released vitamin D
component at the binding site of the host component to form a
partner/host complex. Preferably this competing molecule is linked
to a separator component, wherein the separator component
eventually facilitates in separating out and isolating the
partner/host complex. The separator component may, without
limitation, comprise a peptide, an antibody, a small molecule, a
polymer particle and/or a magnetic particle. The competing molecule
may be linked to the separating component by various means known in
the art. For example, the molecule may be linked to the separator
component through a chemical bond or via an antibody.
In one embodiment, the separator component may be isolated from
solution, along with anything else which is presently attached to
it, for example a competing molecule, by various means. For
example, if the separating component is an antibody, an
Enzyme-Linked-ImmunoSorbent Assay (ELISA) technique or the like may
be employed to isolate the separating component. If the separating
component is a magnetic particle, then a magnetic field may be
employed to isolate the separating component from solution.
In one embodiment, the partner component comprises a vitamin D
component linked to a solid phase, for example a particle,
preferably a magnetic particle. In a preferred embodiment, the
vitamin D component directly links to a particle. For example, a
3-hydroxyl of the 25-OH-D (a vitamin D component) may form a direct
covalent bond with a functional group present on a particle; such
group includes, without limitation, --COOH, --NH2, epoxide, tosyl,
and --SH. In a more preferred embodiment, the vitamin D component
links to a particle through at least one linking component, for
example a biotin, derivatives thereof and the like. (see Holick et
al U.S. Pat. No. 5,981,779, the disclosure of which is incorporated
in its entirety herein by reference). For example, in one
embodiment, the partner component comprises a 25-OH-D linked to a
magnetic particle via a biotin.
In one embodiment, a partner component competes with the vitamin D
component, for example, the released 25-OH-D, to bind to the host
component, for example an antibody, to form a partner/host complex.
The partner/host is then isolated and its concentration is
determined. The concentration of the vitamin D component, for
example the 25-OH-D, should be directly proportional to the
concentration of the partner/host complex.
In an alternative embodiment, the partner component comprising a
competing molecule linked to a separator component competes with
the vitamin D component to bind to one or more intermediate binding
components. At least one of the intermediate binding components has
a binding site for a vitamin D component. In this case, the host
component can form a complex with the partner component through an
intermediate binding component. (When the partner component forms
an a complex with a host component through an intermediate
component, a partner/intermediate/host complex is formed. The
partner/intermediate/host complex may sometimes be referred to
herein as the "partner/host" complex for clarity.) For example, in
one embodiment, the partner component comprises a 25-OH-D linked to
a magnetic particle through a biotin, and this partner component
competes with a released 25-OH-D at the binding site of an
intermediate binding component, for example a DBP. After the
partner component binds to the intermediate binding component, the
host component, for example an antibody, may form a complex with
the partner component through the intermediate binding component,
by binding to the intermediate component. In a preferred
embodiment, the host component antibody which binds to a DBP
(hereinafter "anti-DBP") is a rabbit antibody purchased under the
trade name Rabbit Anti-Human Gc-Globulin from DAKO in Denmark.
Furthermore, the host component may be present when the partner
component competes with the vitamin D component to bind to the
intermediate binding component; or the partner/intermediate complex
may be first isolated from solution and the host component is
subsequently introduced to form a partner/intermediate/host
complex.
The concentration of the partner/host complex formed in accordance
with this invention should be proportional to the concentration of
the vitamin D component being measured. (Herein after and
throughout "partner/host complex" may refer to a partner/host
complex or partner/intermediate/host complex.) In one embodiment,
the concentration of the partner/host complex may be measured by
isolating the partner/host complex and measuring the level of the
host component and/or measuring the level of the intermediate
component if the partner component forms a complex with the host
component through an intermediate component. Various methods known
in the art may be adapted and employed to measure the concentration
of the host component and/or the intermediate component.
For example, in one embodiment, the host component and/or the
intermediate component (hereinafter "signal compounds") may be
labeled. Preferably, the signal compound is labeled at a position
where the signal compound may still bind to a partner component and
the label may still be detected. Labels which may be employed in
accordance with this invention include, without limitation,
chemiluminescent molecules, fluorescent molecules, enzymes,
co-enzymes, isotopes and sensitizers.
Chemiluminescent molecules useful in this invention include,
without limitation, luciferin, luminol, pyrogallol, isoluminol,
aequorin, cyclic arylhydrazides, dioxetanes, rhodium chelates
(electrochemiluminescent), oxalate esters, thermochemiluminescent
labels, acridinium and the like. These labels may be attached to a
protein, for example an anti-DBP, with techniques well known in the
art. (See U.S. Pat. No. 5,284,952, the disclosure of which is
incorporated in its entirety herein by reference.) In one
embodiment, a signal compound such as an anti-DBP may be labeled
with an acridinium by employing the methods found in U.S. Pat. No.
5,284,952. (See also U.S. Pat. Nos. 5,110,932 and 5,338,847, the
disclosures of which are incorporated in their entirety herein by
reference.) In this embodiment, the acridinium labels the lysine
residue of the anti-DBP. It has been shown that a sufficient number
of the anti-DBP is labeled at the appropriate places to be
effective as a labeled host component.
Fluorescent molecules useful in this invention include, without
limitation, umbelliferone, fluorescein, rhodamine, Texas red dyes,
pthalocyanines, coumarin, squaraine, anthracene, erythrosine,
europium chelates and the like. For example, a signal compound such
as a DBP may be labeled with an umbelliferone. (See also, U.S. Pat.
No. 3,901,654, the disclosure of which is incorporated in its
entirety by reference herein.)
Enzymes useful in this invention include enzymes capable of
catalyzing a reactant to produce a product, wherein the product can
be detected. Exemplary enzymes which have been developed and used
in an assay are those described in U.S. Pat. Nos. 3,654,090;
3,791,932; 3,839,153; 3,850,752; U.S. Pat. Nos. 3,817,837;
3,879,262; Journal of Immunological Methods 1: 247(1972); and the
Journal of Immunology 109:129(1972), the disclosures of which are
incorporated in their entirety herein by reference. Other enzymes
which may be used in accordance with this invention includes
glactsidase, gluconidase, phosphatase, peptidase, alkaline
phosphatase and the like. In one embodiment, an enzyme in
accordance with this invention may be able to catalyze a
chemiluminescent reactant, wherein one of the resulting product is
a light signal, which can be detected.
Co-enzymes useful in this invention include molecules and/or
proteins which facilitate an enzyme to catalyze a reactant to
produce a detectable product, for example light. A co-enzyme may
include, without limitation, FAD and NAD. In one embodiment, an
anti-DBP may be labeled with a NAD. (See for example, U.S. Pat. No.
4,380,580, the disclosure of which is incorporated in its entirety
herein by reference.)
Isotopes useful in this invention include, without limitation,
H.sup.3, p.sup.32 and F.sup.18. Other labels may include a
non-active precursor of a spectrophotometrically-active substance
(British Pat. No. 1,392,403 and French Pat. No. 2,201,299, which
patents correspond to U.S. Pat. No. 3,880,934) and electron spin
resonance moieties (U.S. Pat. No. 3,850,578).
Sensitizers useful in this invention include, without limitation,
those disclosed in U.S. Pat. No. 5,705,622, the disclosure of which
is incorporated in its entirety herein by reference. For example,
an acceptor molecule may be attached to a DBP and an acceptor
molecule may be attached to a vitamin D component.
Various kits comprising a releasing composition and a detecting
composition as disclosed in this invention may be used for
determining the concentration of a vitamin D component. However,
such are only examples of the many possible kits which may employ a
releasing composition to assay for a vitamin D component. Other
kits are contemplated to be within the scope of this invention. For
example, the following patents, articles and instruction manuals
disclose assay methods which may be adapted to include a releasing
composition of the present invention: U.S. Pat. No. 4,935,339
(discloses a delayed capture assay); U.S. Pat. No. 4,121,1978; U.S.
Pat. No. 5,232,836; U.S. Pat. No. 5,064,770; U.S. Pat. No.
5,202,266; U.S. Pat. No. 4,816,417; U.S. Pat. No. 5,821,020 and
U.S. Pat. No. 5,981,779; Competitive Protein-Binding Radioassays
for 25-OH-D by Haddad; Instruction Manual of Jummun Diagnostik
Entitled 25-OH Vitamin D Enzyme-based Protein-Binding-Assay;
Instruction Manual of Incstar Catalog No. 68100; Directional insert
of Nichols Institute entitled 25-OH-D 60T Kit; Directional Insert
of BioSource Europe S.A. entitled 250H-VIT.D3-RIA-CT; Directional
Insert of IDS, Inc. entitled Gamma-B25-Hydroxy Vitamin D RIA
AA-35PS (Jan. 6, 1999, issue 3); Journal of Pharmaceutical and
Biomedical Analysis 20 (1999); Determination of Vitamin D3
metabolites/state-of-the-art by Luque de Castro, et al.; Clin.
Chem. 30/11, 1731 1736 (1984); Two Direct (Nonchromatographic)
Assays for 25-hydroxyvitamin D by Bouillon, et al. The disclosures
of which are incorporated in their entirety by reference herein.
One adaptation would be to replace the compositions used therein
with the present releasing composition.
Without limitation, Table I identifies some general assays (A
through H) in which a releasing composition of this invention may
be employed. Assay A has been discussed above.
Assay B may similarly employ a releasing composition to release the
vitamin D component of a body fluid sample. Once released, it may
be added to a composition or a series of compositions comprising a
labeled DBP and a vitamin D component linked to a separator
component, for example a magnetic particle. Preferably, the
released vitamin D component does not have to be purified prior to
adding to said composition. In other words, a composition
comprising a labeled DBP and a vitamin D component linked to a
separator component may be added to the homogeneous mixture of body
fluid sample and releasing composition.
Assays C through G may similarly employ a releasing composition to
release the vitamin D component from a body fluid sample. Once
released, a detecting composition or series of detecting
compositions, comprising the components identified in the
respective assays, may be added. In assay G, the conditions
conducive for the binding of the biotin to the avidin may be
similar to that disclosed in U.S. Pat. No. 5,395,938, (Title:
Biotinylated Chemiluminescent Labels and Their Conjugates, Assays
and Assay Kits), the disclosure of which is incorporated in its
entirety herein by reference.
Assay H may also employ a releasing composition of the present
invention to release the vitamin D component from a body fluid
sample. After it is released the vitamin D component is allowed to
bind directly to a host component, for example an antibody. The
released vitamin D component is also allowed to bind to a labeled
antibody. Preferably the host component and the labeled antibody
are able to bind to different determinants of the released vitamin
D component. (See U.S. Pat. No. 5,641,690, which discloses
immunometric assay methods, the disclosures of which are
incorporated in its entirety herein by reference). The
host/vitamin-D/labeled antibody complex may be isolated and counted
utilizing techniques commonly known in the art, for example
ELISA.
In a broad embodiment, the kit of the present invention may be
adapted to be employed in an automated assay system to determine
the concentration of a vitamin D component. For example, the kit of
the present invention may preferably be used in conjunction with
the Nichols Advantage system.
EXAMPLE 1
An Automated Method for Detecting a 25-OH-D
Currently, immunoassays measuring the concentration of 25-OH-D use
organic solvents to release the 25-OH-D. The addition of an organic
solvent to the serum results in either a precipitate or a two-layer
liquid. Thereafter, the layer containing the released 25-OH-D is
(manually) separated and is mixed with another composition to
measure the concentration of the 25-OH-D. The step of separating
the 25-OH-D is (a) time consuming, and (b) a source of error in the
assays, especially if the separation step is performed
manually.
The present invention eliminates the need to purify or
substantially purify the released 25-OH-D prior to determining its
concentration and allows for automation of the assay. In this
example, the automated release of the 25-OH-D from a binding
protein and determination of the 25-OH-D concentration are
performed on the Nichols Advantage system. This process is wholly
automated. Manual pipetting and centrifugation are not needed. The
assay kit use in conjunction with the Nichols Advantage System
includes a release composition and detecting composition.
Releasing Composition
The releasing composition comprises an aqueous base component (0.1
1.0 M NaOH), beta-randomly methylated cyclodextrin (0.01 5%), and
sodium salicylate (0.01 5%). The pH of the releasing composition is
about 13.
Detecting Composition
The detecting composition comprises a magnetic particle suspension
with 25-OH-D immobilized on the surface, a DBP, an anti-DBP and
stabilizers. Also, the detecting composition has a pH of about 6 to
about 9, preferably 8.3. Furthermore, the detecting composition
also has a buffer capacity to bring the pH of the total mixture
(the homogeneous mixture and the detecting composition) into the
range where the DBP is stable and capable of binding 25-OH-D.
Preferably the total mixture has a pH of about 6 to about 9,
preferably 8.3. The buffer system used comprises 200 mM TRIS, 150
mM NaCl, 0.9% gelatin, 1% bovine serum albumin and 0.09% sodium
azide.
The streptavidin coated particles are about 0.5 5 microns in size,
and may be purchased from Seradyne, Dynal, Bangs Laboratories. The
25-OH-D is attached to the particle via a biotin or the like
(Holick et al U.S. Pat. No. 5,981,779, the disclosure of which is
incorporated in its entirety herein by reference). The amount of
particles used in the assay is about 5 to about 30 uL (preferably
25 uL) of a 1.25 2.5 mg/mL suspension.
The concentration of the DBP is in the range about 0.2 to about 1.0
ug/mL and is added to the assay in the amount of about 10 to about
280 uL, preferably about 250 uL.
The anti-DBP, preferably purchased from DAKO, is labeled with an
acridinium sulfonyl chloride derivative, a chemiluminescent label.
Preferably, the acridinium derivative is labeled by a method
disclosed in U.S. Pat. No. 5,284,952, the disclosure of which is
incorporated in its entirety herein by reference. In brief, the
conjugate is prepared by adding the reactive label in acetonitrile
to the antibody in carbonate buffer at pH 9.6. The reaction
proceeds at ambient temperature for a period of about half an hour
and the unattached label is removed by gel filtration
chromatography. The labeled anti-DBP is diluted to a concentration
of about 0.2 10 ug/mL in buffer with a pH of about 6.0. The amount
of acridinium labeled anti-DBP that is added to the assay is about
10 to about 280 uL, preferably about 50 uL.
In preferred embodiments, a stabilizer may be added to a
composition of the assay. For example, a stabilizer may be added to
the detecting composition. Stabilizers include gelatin, bovine
serum albumin, egg albumin, polyethylene glycol, poly vinyl
alcohol, poly vinyl pyrrolidone and the like and mixtures thereof.
These stabilizers may be effective in blocking of the non-specific
binding of the assay components.
About 10 100 uL, preferably 20 uL, of a patient body fluid sample
and about 10 100 uL, preferably 60 uL, of the releasing composition
are pipetted into a holder, for example a cuvette well, forming a
homogeneous mixture. The releasing composition acts on the patient
sample in an incubator at a constant temperature (about 25
37.degree. C., preferably 37.degree. C.) for a specified time (1 60
min., preferably 21 min.). When the release is complete, a
detecting component is pipetted directly into the homogeneous
mixture, forming a total mixture. The total mixture is incubated
for about 30 min to about 60 min., preferably about 42 min. During
this incubation period the 25-OH-D-biotin-magnetic-particle
competes with the 25-OH-D of the patient's sample to form a complex
with the labeled-DBP. Subsequently, the
25-OH-D-biotin-magnetic-particle/labeled-DBP complex is isolated by
a wash. The concentration of the complex is determined by a
chemiluminescent reading. The concentration of the complex is
inversely proportional to the concentration of the 25-OH-D
concentration in the patient's sample.
In addition to decreasing the labor requirements, this assay method
allows for positive patient identification. Positive patient
identification can be achieved because the Nichols Advantage system
pipettes the detecting composition directly into the sample holder
containing the releasing composition and the body fluid, and every
sample holder has a bar code to which the result matches.
EXAMPLE 2
Precision of Assays: Intra CV
Intra Coefficient of Variation (CV) studies for the determination
of the 25-OH-D were conducted with various systems, including a
system using a kit of the present invention in conjunction with
Nichols automated assay machine, the Nichols Advantage. The studies
using the kit of the present invention and Nichols Advantage
automated technique involves obtaining a patient sample and
performing the assay 20 times. The results are shown in Table
I.
TABLE-US-00001 TABLE I Dose (ng/ml) CVD Nichols 12.3 7.7% Advantage
34.6 3.5% Automated assay 86.0 2.0% Run #1 Nichols 9.5 14.1%
Advantage 46.8 2.6% Automated assay 58.6 2.6% Run #2 Dose CVD
.sup.125I Assay Kit #1 26.5 5.3% 58.4 5.0% 151.0 6.1% .sup.125I
Assay Kit #2 8.6 11.7% 22.7 10.5% 33.0 8.6% 49.0 12.5%
Each patient sample was assayed 20 times. Of the 20 results, a mean
and a standard of deviation were determined. CV=(standard of
deviation/mean).times.100. CVD is CV of Dose, wherein Dose is the
concentration of the 25-OH-D. The lower the CV, the better the
precision of measurement. For example, a good CVD is <8%.
For each Dose range, the CVDs of the assays using an assay kit of
the present invention in conjunction with the Nichols Advantage
system are better than other assay systems. For example, the assay
kit of the present invention has a CVD of 2.6% at a Dose of 46.8;
the .sup.125I Assay Kit #2 has a CVD of 12.5% at a Dose of 49.0;
and the .sup.125I Assay Kit #1 has a CVD of 5.0% at a Dose of
58.4.
EXAMPLE 3
Luminol Chemiluminescent Direct Labeling of DBP
About 1 mg of Vitamin D binding protein, DBP, is buffer exchanged
three times with 100 mM PBS at pH 8.2. The final volume was 200 uL.
Succinyl-amino-buty-ethyl-isoluminol NHS ester (4.6 mg) is
dissolved in DMF. The isoluminol (17 uL) is added to the DBP and
the mixture is maintained at room temperature for about 1 hour with
occasional shaking. The unreacted label is removed by size
exclusion chromatography using SEPHADEX-G25 (23.times.1 cm) and 100
mM PBS, pH 6.0 as the mobile phase.
EXAMPLE 4
Direct Chemiluminescent Direct Labeling of DBP
About 1 mg of Vitamin D binding protein, DBP, is buffer exchanged
three times with 20 mM sodium carbonate buffer at pH 9.6. The final
volume is 500 uL. Two aliquots of fluorescein-5-isothiocyanate
(FITC) are dissolved in DMF just prior to addition to give a
concentration of 1.25 mg/mL. The first FITC aliquot (5 uL) is added
and the mixture is allowed to react at room temperature. After 15
minutes the second aliquot (5 uL) is added and the reaction
proceeds for an additional 15 minutes. The unreacted label is
removed by size exclusion chromatography using Sephadex G-25 (2
cm.times.33 cm) and 100 mM, pH 7.4 mobile phase.
EXAMPLE 5
Biotin Direct Labeling of DBP for "Upside-Down" Assay
About 2.0 mg of DBP is buffer exchanged with 20 mM bicarbonate at
pH 9.6 three times to give a final volume of 1.0 mL. Two aliquots
of sulfo-NHS-LC-biotin (Pierce) are dissolved in water to give a 1
mg/mL solution just prior to addition. The first aliquot (10 uL) is
added and the reaction is maintained at room temperature for 5
minutes. The second aliquot (10 uL) is added and the reaction
proceeds for an additional 4 minutes. The unreacted biotin is
removed by size exclusion chromatography using SEPHADEX G-25 and
100 mM PBS, pH 7.4 as the mobile phase.
EXAMPLE 6
Acridinium Direct Labeling of anti-25-Hydoxyvitamin D Monoclonal
Antibody
2.5 mg of antibody is buffer exchanged into 20 mM bicarbonate
buffer, pH 9.6, three times to give a final volume of 1.75 mL.
Sulfonylchloride acridinium ester is dissolved in sufficient
acetonitrile to give a 1.75 mg/mL solution. The acridinium ester
(52.5 uL) is added to the antibody and the reaction mixture is
maintained at room temperature about 0.5 hour. The unreacted label
is removed by size exclusion chromatography using SEPHADEX G-75 and
100 mM PBS, pH 6.0 as the mobile phase.
EXAMPLE 7
Acrdinium Indirect Labeling
Anti-DBP (Dako) is buffer exchanged to yield 2.2 mg of protein in
1.1 mL of pH 9.6, 20 mM bicarbonate buffer. A solution of
sulfonylchloride acridinium ester (1.6 mg/mL) is in acetonitrile.
The acridinium (4.4 uL) is added to the antibody and the reaction
mixture is maintained at room temperature for 15 minutes. A second
aliquot of acridinium (4.4) uL is added for and additional 15
minutes. The unreacted label is removed by chromatography on a
mixed bed column (SEPHAROSE 6B/SEPHADEX G-75, 1.5 cm.times.48
cm).
EXAMPLE 8
Direct Labeling of DBP with HRP
Activated peroxidase from Boehringer Mannheim Biochimica is used
for labeling. The DBP (4 mg) is dissolved in pH 9.8, 100 mM
carbonate buffer (1 mL). This solution is added to the POD (8 mg).
The reaction proceeded at room temperature for 1 hour. The
conjugation is quenched by addition of 120 uL of triethanolamine
solution (pH 8, 30%). Sodium borohydride (150 uL) freshly dissolved
in water (8 mg/mL) is added to the solution maintained at room
temperature for about 0.5 hour. Additional sodium borohydride (75
uL) is provided and the reaction proceed for an additional 2 hours
at room temperature. The conjugate is dialyzed with pH 6.0 PBS with
glycine.
While this invention has been described with respect to various
specific examples and embodiments, it is to be understood that the
invention is not limited thereto and that it can be variously
practiced with the scope of the following claims.
* * * * *